Abstract
NF-kappa B essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappa B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappa B activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappa B activation was quantified using an NF-kappa B-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappa B activation with an IC(50) in the mum range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappa B activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappa B pathway activation through targeting the oligomerization state of the NEMO protein.
Highlights
Nuclear factor-B (NF-B)1 signaling is a transduction pathway involved in a variety of essential cellular processes includ
Rational Design of Peptides from the Minimal Oligomerization Domain of NEMO—We have identified the minimal oligomerization domain required for the formation of the NEMO trimer
Our model predicts that the Coiled-coil 2 (CC2)-leucine zipper (LZ) domain forms a six-stranded helical bundle composed of a trimer of CC2 domains surrounded by three LZ coiled-coils [16]
Summary
Nuclear factor-B (NF-B) signaling is a transduction pathway involved in a variety of essential cellular processes includ-. The N-terminal part of the polypeptide contains a large coiled-coil motif (CC1) and all the residues involved in the interaction of the protein with the IKK kinases (IKK-binding domain, see Fig. 1) [13]. The C-terminal half (residues 250 – 412) is composed of two successive coiledcoil motifs, CC2 (residues 253–285) and LZ (residues 301–337), and a zinc finger motif (ZF) at the extreme C terminus of the polypeptide It is this C-terminal part of NEMO that is involved in the oligomerization of the protein [14, 15]. The C-terminal region is essential for protein function as NF-B activation in NEMO-deficient cells cannot be restored by complementation with a gene carrying a mutation in the oligomerization domain [16]. Therapeutic peptides have already been successfully designed, such as the gp peptide involved in HIV-1 penetration into cells [24, 25]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
